Proteins with a CAAX motif regulate a number of pathways important in oncogenesis. These proteins undergo a series of post-translational modifications that are important for their localization, stability and function. The modifications are initiated by the addition of an isoprenoid moiety (farnesyl or geranylgeranyl) to the cysteine of the CAAX motif by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase-1 (GGTase-1) respectively. This is followed by the endoproteolytic release of the terminal tripeptide (AAX) by RAS converting enzyme (RCE1) and carboxylmethylation of the C-terminal prenylcysteine by isoprenylcysteine carboxyl methyltransferase (Icmt) (FIG. 1).
The most widely studied example of CAAX proteins is the RAS family of regulatory proteins. RAS is a very important molecular switch for a variety of signaling pathways that control diverse processes like cytoskeletal integrity, proliferation, cell adhesion, apoptosis and cell migration. Activating mutations in RAS genes are implicated in the pathogenesis of a large number of solid tumors and hematologic malignancies. In addition, many cancers contain alterations upstream of RAS in signaling cascades and the resultant hyperactivation of RAS is thought to contribute to tumorigenesis.
The possibility of blocking RAS-induced oncogenic transformation by inhibiting the enzymes involved in the post-translational processing of the CAAX motif has been explored for its therapeutic potential. The protein prenyltransferases in particular FTase have been targets of major drug discovery programs. FTase inhibitors showed significant activity in mouse models but clinical trials in cancer patients were disappointing, possibly due to the geranylgeranylation of substrates by GGTase1 when FTase was inhibited. Hence, attention has shifted to the post-prenylation enzymes RCE1 and Icmt as potential therapeutic targets. In particular, there is keen interest in developing Icmt inhibitors in view of studies that showed that genetic and pharmacological intervention with Icmt activity led to significant impairment of oncogenesis in several tumor cell models.
To date, four broad classes of Icmt inhibitors have been investigated. The first class comprises S-adenosylhomocysteine (AdoHcy) and compounds that increase intracellular AdoHcy. AdoHcy is formed when a methyltransferase catalyzes the transfer of the methyl group from S-adenosylmethonine (AdoMet) to the substrate. AdoHcy binds to and competitively inhibits methyltransferase activity. However, AdoHcy is not a selective inhibitor of Icmt and affects the activity of other cellular methyltransferases.
The second class of ICMT inhibitors is structural analogues of the substrate prenylcysteine. Examples are N-acetyl-S-farnesyl-L-cysteine (AFC) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC). These compounds are competitive inhibitors of Icmt but as structural mimics of the carboxy-terminal prenylcysteine of processed CAAX proteins, they can be expected to affect a large number of processes controlled by CAAX proteins. The more potent analogs identified through these studies are depicted in FIG. 2. Replacement of the amide bond in AFC with the metabolically stable and more drug-like sulphonamide linkage gave A which inhibited Icmt with an IC50 of 8.8 μM when evaluated on a vapour diffusion assay. The allylic thioether is deemed to be undesirable due to its chemical and enzymatic lability, thus prompting its replacement with a triazole moiety. The most potent triazole prenyl cysteine analog B has an IC50 of 19.4 μM. Another modification involved replacing two of the isoprenoid units in the farnesyl side chain of AFC with an aryl alkyl moiety. C was identified as the most potent analog in that investigation.
The third category comprises small molecule inhibitors of Icmt. The first compound to be identified was cysmethynil (2-[5-(3-methylphenyl)-1-octyl-1H-indolo-3-yl]acetamide) which was discovered through the screening of a diverse chemical library made up of over 70 sub-families with unique scaffolds.

It is a competitive inhibitor of the isopenylated cysteine substrate and a non-competitive inhibitor of the methyl donor AdoMet. Inhibition is time dependent and involves the formation of an initial reversible complex with the enzyme (Ki 2.39 μM), followed by a conformational change to give a tighter EI* complex with an overall dissociation constant of 0.11 μM. Cysmethynil caused the mislocation of RAS and impaired epidermal growth factor signaling in cancer cells. It blocked anchorage-independent growth in a colon cancer cell line which was reversed by overexpression of Icmt. Cysmethynil was reported to induce autophagic cell death.
Cysmethynil is poorly soluble and binds strongly to plasma proteins. A quick assessment of its compliance to drug-like filters like the Lipinski's “Rule of Five” and other criteria shows that it exceeds the lipophilic threshold for drug-likeness (Estimated Log P of cysmethynil is 7) and just complies with the cut-off value for rotatable bonds.
In 2011, Judd and co-workers (Judd W R et al. 2011. J Med Chem 54, 5031) investigated the Icmt inhibitory potential of methylated tetrahydropyranyl derivatives and reported 3-methoxy-N-[2-(2,2,6,6-tetramethyl-4-phenyltetrahydropyran-4-yl)ethyl]aniline

3-methoxy-N-[2-(2,2,6,6-tetramethyl-4-phenyltetrahydro pyran-4-yl)ethyl]aniline as a potent nanomolar inhibitor of Icmt. Icmt inhibition was determined by a fluorometric coupled enzyme assay for SAM-dependent methyl transferase and reconfirmed using the direct radiometric assay which is traditionally used for measuring Icmt inhibition. The compound showed a dose-dependent increase in Ras cytosolic protein and was modestly cytotoxic on several malignant cell lines, irrespective of their Ras status. GI50 values ranged from 0.3 to >100 μM. Interestingly, the authors found that a farnesyltransferase inhibitor FTI-2628 was significantly more potent on cells that harbour Ras mutations than those with wild type Ras. Hence they proposed that the inhibition of the prenylation step of CAAX proteins was more effective in reducing cell viability than inhibition of the Icmt-mediated methylation step with this class of small molecule Icmt inhibitors.
S-Farnesyl-thiosalicylic acid to inhibits both Icmt and H-ras driven cell growth. However it was considered that inhibition of ras dependent cell growth was not related to the inhibition of ras methylation by Icmt.

The last category of compounds comprises a miscellaneous group of natural products that have been found to possess Icmt inhibitory activity. They range from chemical entitites isolated from marine sponges (spermatinamine, aplysamine 6) to plants (prenylated β hydroxychalcones, a flavanone S-glabrol). Most of these compounds are modest inhibitors (IC50>10 μM) and lack drug-like features.